1. Technical Field
The present invention relates to a method of separating free-astaxanthin selectively from green algae and, more particularly, to a method of separating free-astaxanthin selectively from a green microalgae Haematococcus pluvialis. 
2. Discussion of Related Art
Red ketocarotenoid astaxanthin is a kind of carotenoid pigment having the same chemical structure as β-carotene, and is an anti-oxidizing material having an ability of removing detrimental activated oxygen that causes aging or a cancer. Astaxanthin has outstandingly higher anti-oxidizing activity than that of an existing anti-oxidizing material since it has a unique molecular structure having one more a hydroxyl group (—OH) and a ketone group (═O) at both end groups compared to β-carotene. The anti-oxidizing activity of astaxanthin is about 500-fold higher than representative anti-oxidizing agent vitamin E, and about 20-fold higher than β-carotene. Due to such high anti-oxidizing activity, astaxanthin is being widely used as a pharmaceutical, a food additive, and a feed additive for an animal and a fry. Further, it is expected that the demand and the application range for astaxanthin be rapidly expanded.
A method of producing astaxanthin includes a chemical synthesis, a method of extracting from shells of the Crustacea such as a crab and a shrimp, and a direct synthesis employing microorganism strains Phaffia rhodozyma and Haematococcus pluvialis. 
The astaxanthin synthesized by chemical synthesis has less in vivo absorption rate than that of natural one, has a problem in safety as a food additive, and thus FDA approval was not given to it as a food additive. Only some countries such as Norway, Chile and Canada approve it as a food additive.
Following research results that natural astaxanthin is deposited on a living organism in high concentration compared to synthetic astaxanthin, natural astaxanthin have been produced from byproducts of a shrimp or a crab, however, there is a problem that the natural astaxanthin thus produced is not suitable as an astaxanthin source due to the difficulty in its separation and purification process.
Consequently, biosynthesis through strain cell culture is required for producing natural astaxanthin. Strains that can be used industrially include P. rhodozyma and H. pluvialis. Green algae H. pluvialis has problems of low growth rate, thick cell wall and low cell density, even though it has outstandingly high amount of accumulated astaxanthin compared to yeast P. rhodozyma. Therefore, H. pluvialis is behind P. rhodozyma having high growth rate in preference and industrial applicability, but is deemed to be the most promising strain since 3S, 3S′ astaxanthin isomer that only Haematococcus species possess is assumed to elevate the stability at lipid environment. Accordingly, for industrial application for astaxanthin by Haematococcus species, not only relevant techniques for cell culture in high concentration and for producing astaxanthin in high concentration, but also techniques for recovering the astaxanthin accumulated in a cell with high efficiency must be developed. Particularly, the techniques for culturing photosynthetic microalgae in high concentration have been actively developed through development of photo incubator, etc., but the techniques for separating Haematococcus astaxanthin in high concentration have not been actively developed. Therefore, a method of obtaining astaxanthin extract with high purity has not been developed for industrial scale.
A method of recovering the representative Haematococcus astaxanthin proposed hitherto includes a mechanical method of extracting astaxanthin by freezing a strain from which water is completely removed with liquid nitrogen, then pulverizing it with an impact mill, and then disrupting the cell wall of the strain; a physical method of extracting astaxanthin with a solvent after disrupting the cell wall of the strain with a machine such as a physical homogenizer; a biological method of extracting astaxanthin after disrupting the cell wall of the strain by employing an enzyme disrupting a cell wall, i.e., cellulase, pectinase, protease, etc. However, these methods have problems of low extraction speed, high disruption rate for astaxanthin, much labor force and high process cost, etc., and thus are not put to practical use. Accordingly, for industrial mass production of astaxanthin employing Haematococcus species, there is needed for a new process of separating astaxanthin capable of recovering astaxanthin in high concentration from a strain with high efficiency.